The invention relates to the field of bioconversion, using plant enzymes for the production of flavor, fragrance, pharmaceutical or bio-control agents from less valuable substrates. More specifically it relates to a process for production of terpenoid compounds by the use of plant enzymes.
Modern chemistry strongly depends on the use of catalysts in order to have efficient and clean reactions with a minimum of waste. Especially the use of biocatalysts (enzymes, cells) is strongly increasing, also in industry. The most important features of biocatalysts are (Faber, 2000):                1. they are very efficient;        2. they are environmentally acceptable;        3. they operate under mild conditions;        4. they are selective.        
Up to now, the large majority of enzymes used in bioconversion employing biocatalysis for industrial and laboratory applications is obtained from microbial sources. A minor fraction of enzymes is obtained from plant sources (Faber, 2000). Nevertheless, the plant kingdom is an important source for the chemist and the biotechnologist because plants produce a unique variety of chemicals (Franssen and Walton, 1999). The rationale for this is simple: there is no way plants can escape from their predators, so they have to defend themselves by chemical ways. Plants make compounds with the most fantastic chemical structures: the antitumor drug TAXOL® (paclitaxel) (from Taxus brevifolia), the insect feeding deterrent azadirachtin (from the Indian neem tree Azadirachta indica) and the analgesic morphine (from Papaver somniferum). It is mainly because of the tremendous importance of compounds like these that science has been, and still is, interested in the application of plant cells and enzymes, irrespective of the disadvantages they sometimes have (Walton and Brown, 1999). By far the most important group of secondary metabolites, containing a vast number of components that act as flavor, fragrance, pharmaceutical or bioactive (insecticidal, anti-microbial, repellent, attractant, etc, etc) compounds, are the terpenoids. The terpenoids belong to the isoprenoids. By definition isoprenoids are made up of so-called isoprene (C5) units. This can be recognized in the number of C-atoms present in the isoprenoids which usually can be divided by five (C5, C10, C15, C20, C25, C30 and C40), although also irregular isoprenoids (e.g. C13, C16) and polyterpenes (Cn) have been reported.
The terpenoids consist of a.o. monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes and polyterpenes (rubbers), etc. Mono- and sesquiterpenes, the C10 and C15 branch of the isoprenoid family, are economically interesting as flavor and fragrance compounds in foods and cosmetics, and can have anti-carcinogenic effects and antimicrobial properties. Mono- and sesquiterpenes have also been shown to be of ecological significance, for instance in the interaction and signaling between plants, plants and insects/spider mites and plants and microorganisms.
The sesquiterpene lactones are an important subgroup of the sesquiterpenes, and over 4000 differentstructures have been identified. A wealth of information is available about the structural aspects and biological activities of these type of compounds (e.g. Picman, 1986; Harborne et al., 1999; Seigler, 1995).
Sesquiterpene lactones may constitute up to 5% of the dry weight of the plant and occur mainly in members of the Asteraceae (in about 450 species), the largest of all plant families, but also occur in other higher plant families such as the Apiaceae (12 species) and lower plants such as liverworts, for examnple the genus Frullania (Harborne et al., 1999). Sesquiterpene lactones may occur throughout a plant, but are most commonly associated with leaves, flower parts and taproots.
The Asteraceae contain 1317 genera and 21000 species. They are mainly herbaceous plants, sometimes shrubs or trees, usually with a taproot, sometimes with tubers. Many of the Asteraceae contain (essential) oil bearing organs, such as ducts or trichomes, some of them contain latex. The Asteraceae contain many economic and ornamental plants, such as Sunflower (Helianthus annuus; for the production of seeds and sunflower oil), Jerusalem-Artichoke (Helianthus tuberosus; of which the tubers are eaten and that is also used for the fructan production), and Artemisia annua (for the production of the effective anti-malarial artemisinin. Also used as food are the roots of Salsify (Tragopogon porrifolius) and Scorzonera (Scoizonera hispanica), the young flower-heads of Globe Artichoke (Cynara scolymus), and the leaves of Lettuce (Lactuca sativa), Endive (Cichorium endiva) and Radicchio and Brussels Endive (Cichorium intybus). In addition, there are many ornamental Asteraceae, for example in the genera Dahlia, Doronlicum, Heleniumn, Tagetes, Helianthus, Aster, Centaurea, Gerbera, etc.
The occurrence of sesquiterpene lactones in the tribes of the Asteraceae is of taxonomic interest. They are common in the Heliantheae, occur frequently in the Anthemideae, Cichorieae; Cynareae, Senecioneae, Inuleae and Vernonieae, only infrequently in the Eupatoriaea and not in the Astereae, Mutisieae, Tageteae and Arcoteae-Calenduleae (Seigler, 1995). Crop species that have been shown to contain sesquiterpene lactones are for example: Artichoke, Lettuce, Endive, Radicchio, Brussels Endive, Sunflower, Jerusalem Artichoke, Artemisia annua, Matricaria recutita. Some wild (or ornamental) Asteracea species containing sesquiterpene lactones are: Lactuca virosa, Achillea spp., Ambrosia spp. Cnicus benedictus, Artemisia spp., Xanthium spp., Iva axillaris, Parthenium spp., Helenium spp., Hymenoxys odorata, Vernonia spp., Vanillosmopsis spp., Eremanthus spp., Moquinea spp., Parthenium spp., Arnica spp., Atractylodis macrocephala, Eupatorium cannabium, Achillea millefolium, Tanacetum (Chrysanthemum) vulgare, Inula helenium and Taraxacum officinale (Seigler, 1995; Van Genderen et al., 1996).
One example of an Asteraceous species that contains large amounts of sesquiterpene lactonesis Chicory. The roots of chicory are extremely bitter, due to these sesquiterpene lactones.
Chicory is a blue-flowered composite plant that has spread all over the world from the Mediterranean and east Asia. Since the seventeenth century it has been cultivated (var. sativum) for its bitter roots that were roasted and used in hot ‘coffee-like’ beverages. While the use of its roots was displaced by genuine coffee from Coffea arabica, sprouts of chicory (var. foliosum) that are grown in the dark became popular as a vegetable (Belgian endive) halfway through the nineteenth century. Nowadays it is a common crop in Belgium, northern France and The Netherlands (Weeda et al., 1991; Vogel et al., 1996). During the first year of growth, the plant develops a deep taproot and produces a rosette of leaves on a short stem. Following a period of cold exposure, the plant develops a floral meristem. Commercial production involves harvesting the plant following the attainment of a proper stage of maturity of the root, followed by floral bud induction (cold storage) and then an accelerated but controlled development of the floral axis and surrounding basal leaves in the dark (forcing). The end product of forcing is a chicon, a small white head of leaves ringed with regions of yellow-green. This Belgian endive is known for its slightly bitter taste originating from sesquiterpene lactones (Pyrek, 1985; Seto et al., 1988; van Beek et al., 1990; Price et al., 1990). The major sesquiterpene lactones of chicory are guaianolides, but smaller amounts of eudesmanolides and germacranolides are also present. The bitter taste of chicory is in particular associated with the presence of the guaianolides lactucin, 8-deoxylactucin, and lactucopicrin.
Although a lot of information is available about the structural aspects and biological activities of the sesquiterpene lactones, little is known about their biosynthesis. By far the largest group of naturally occurring sesquiterpene lactones is the group of germacranolides, and the majority of sesquiterpene lactones are thought to evolve from this group. The simplest member of the germacranolides, (+)-costunolide, is generally accepted as the common intermediate of all germacranolide-derived lactones (Geissman, 1973; Herz, 1977; Fischer et al., 1979; Seaman, 1982; Song et al., 1995).
(+)-Costunolide was first isolated from costus roots (Saussurea lappa Clarke) by Paul et al. (1960) and Somasekar Rao et al. (1960), and has since been found with other sesquiterpene lactones in various plants (Fischer et al., 1979). Amongst them is lettuce (Lactuca sativa), a species that is closely related to chicory and also contains the bitter tasting compounds lactucin and lactucopicrin (Takasugi et al, 1985; Price et al., 1990).
Recently we have demonstrated that the sesquiterpenoid backbone of the sesquiterpene lactones in chicory is formed by a (+)-germacrene A synthase which cyclizes FPP to (+)-germacrene A (de Kraker et al., 1998; Bouwmeester et al., 1999b). This (+)-germacrene A is not further transformed into a guaiane or a eudesmane skeleton, indicating that functionalization of the molecule precedes its cyclization. Studies on the biosynthesis of santonin (Barton et al., 1968) suggested that lactone formation precedes any other oxidation of the sesquiterpenoid ring system (Cordell, 1976), and various authors have proposed a biosynthetic route from (+)-germacrene A (8) toward (+)-costunolidce ((Geissman, 1973; Herz, 1977; Seaman, 1982; Fischer, 1990; Song et al., 1995). In this hypothetical route (+)-germacrene A is hydroxylated to germacra-1(10),4,11(13)trien-12-ol that is further oxidized via germacra-1(10),4,11(13)-trien-12-al to germacra-1(10),4,11(i3)-trien-12-oic acid. The germacrene acid is thought to be hydroxylated at the C6-position andsubsequent loss of water leads to the formation of a lactone ring such as present in (+)-costunolide.
Unfortunately, germacrenes are notoriously unstable compounds, susceptible to proton-induced cyclizations and heat induced (e.g. steam distillation, GC-analysis) Cope rearrangement (Takeda, 1974; Bohlman et al., 1983; Reichardt et al., 1988; Teisseire, 1994; de Kraker et al, 1998). None of the intermediates between (+)-germacrene A and (+)-costunolide has ever been isolated, apart from germacra-1(10),4,11(13)-trien-12-al that was isolated with greatest difficulty from Vernonia glabra and could not be separated from its cyclization product costal (Bohlman et al, 1983). Probably as a result of this instability, the hypothetical biosynthetic route for (+)-costunolide has merely been based on the isolation from costus roots of the Cope-rearrangement products (−)-elema-1,3,11(13)-trien-12-ol and (−)-elema-1,3,11(13)-trien-12-al, and the proton-induced cyclization products costol, costal and costic acid (Bawdekar and Kelkar, 1965; Bawdekar et al., 1967; Maurer and Grieder, 1977).
The next step in the biosynthesis of sesquiterpene lactones is catalysed by a cytochrome P450 enzyme (D)e Kraker et al., 2001). Cytochrome P450 enzymes (Mr=±50,000) mostly catalyzes oxidation reactions, but also reductions. Most vertebrate genomes contain more than 30 different structural genes for cytochromes P450 (Mathews and van Holde, 1996), maiing this a large and diverse protein family. These proteins resemble mitochondrial cytochrome oxidase in being able to bind both O2 and CO. Cytochromes P450, however, strongly absorb light at 450 nm when they are in the reduced state and complexed with CO. Light of 450 nm displaces CO from the heme, hence CO binding is photoreversible. For this reason, cytochrome P450 enzymes exhibit photoreversible inhibition by CO (Donaldson and Luster, 1991). Plant cytochrome P450 monooxygenase systems are associated with the endoplasmic reticulum or a prevacuole, and consequently are located in the light membrane (microsomal) fraction of the cell.
Cytochrome P450 enzymes are involved in the hydroxylation of a large variety of compounds. There are two classes of cytochrome P450 activities known: those involved in biosynthetic routes and those involved in detoxification of xenobiotics (Donaldson and Luster, 1991; Mihaliak et al., 1993; Schuler, 1996). These reactions include the hydroxylations of steroid hormone biosynthesis, and the synthesis of hydroxylated fatty acids and fatty acid epoxides. In addition, cytochromes P450 act upon thousands of xenobiotics, including drugs such as phenobarbital and environmental carcinogens such as benzopyrene, a constituent of tobacco smoke. Hydroxylation of foreign substances usually increases their solubility and is a step in their detoxification, or metabolism and excretion.
Cytochrome P450 systems participate in a wide variety of additional reactions, including epoxidation, peroxygenation, desulfuration, dealkylation, deamination, and dehalogenation. These reactions are particularly active in the liver, where a number of cytochromes P450 are xenobiotic-inducible; that is, their synthesis is stimulated by substrates that are metabolized by these enzymes (Mathews and van Holde, 1996). Inducers include drugs such as phenobarbital and other barbiturates.
The amino acid residues that constitute the active site of the enzyme determine the specificity of a given P450. They can vary widely between different cytochromes P450; however, the principal component of the active site of all these enzymes is a heme moiety. The iron ion of the heme moiety is the site of the catalytic reaction, and is also responsible for the strong 450 nm absorption peak in combination with CO. The substrate specificity of cytochrome P450 enzymes depends on their function in the organism: the biosynthesis of metabolites or the breakdown of xenobiotics in animals. The enzymes that take care of the breakdown of xenobiotics have a low specificity, so that a few enzymes can protect the organism against a large diversity of xenobiotics. How the detoxification of xenobiotics in plants is accomplished is not clear yet. Enzymes that are involved in biosynthetic routes of metabolites in plants or animals in general have a very high substrate specificity (Donaldson and Luster, 1991; Mihaliak et al., 1993; Schuler, 1996).
Many different enzymes are involved in the biosynthetic pathways of complex plant metabolites. The enzymes catalyse, among others, stereo- and regioselective hydroxylations, (ep)oxidations, reductions, glycosylations, esterifications and cyclisations. Especially the enzymes that are involved in hydroxylations and oxidations have great potential in chemistry, since the corresponding chemical transformations involve the use of toxic reagents and halogenated solvents, which is undesirable because of safety and environmental reasons (March, 2001). For example, allylic hydroxylations are usually performed using selenium diooxide in methylene chloride (Jerussi 1970). The direct introduction of a carbonyl function next to a carbon-carbon double bond requires the use of toxic chromium trioxide (March, 2001) or the expensive and highly toxic ruthenium tetroxide (e.g., Petit and Furstoss, 1995). Furthermore, hydroxylating and oxidizing enzymes usually have far greater regio- and stereoselectivity than the chemical reagents. A few examples are given below.
Partly on account of their importance in fragrance and flavour industries, the hydroxylation and further biotransformation of terpenoids has been particularly well studied, using cell cultures of essential oil producing species and of other plants, such as Nicotiana tabacum (Suga and Hirata, 1990). Stereospecificity was shown, for example, by the formation of only trans isomers from hydroxylation occurring at C4 of β-terpineol and its acetate and by the predominant formation of a trans-diol as a result of hydroxylation of the endocyclic double bond of α-terpinyl acetate (FIG. 4).
Digitoxin is a cardiac glycoside from Digitalis sp. Its β-hydroxylation on the 11-position yields the more potent digoxin. This has been achieved by purified, reconstituted and immobilized digitoxin 12β-hydroxylase (a cytochrome P450 enzyme complex) from Digitalis lanata in a bioreactor (Petersen and Seitz, 1988; Petersen et al., 1987).
Sesquiterpene alcohols and ketones are an interesting group of compounds for a number of reasons. Sesquiterpene alcohols, for example have been shown to be involved in resistance against micro-organisms: the Solanaceae for example produce sesquiterpene alcohol phytoalexins upon infection with pathogenic fungi and in vitro assays have shown that these sequiterpene alcohols have a strong antifugal activity. Also a number of sesquiterpene alcohols are important in the flavor and fragrance industry, for example santalol, typical for the very expensive sandalwood essential oil, and khusimol, patchoulol, etc. Also the sesquiterpene ketone, nootkatone is a commercially important compound. Because of its excellent organoleptic qualities and in particular its typical grapefruit taste, nootkatone is a widely used ingredient in perfumery and the flavor industry. Nootkatone was also shown to inhibit gastric lesion which explains that it is a constituent of some stomach medications (Yamahara et al., 1990). Although nootkatone can be obtained from valencene or other substrates using chemical synthesis (Hunter and Brogden, 1965; Wilson and Shaw, 1978; Canadian patent no 901601; M. Pesaro et al., 1968; Birch, 1974; U.S. Pat. No. 5,847,226) there is a large demand for natural nootkatone. At present, such a quality can only be obtained by extraction of natural products containing nootkatone, in particular grapefruit, a method which is hardly economic. With the enzymes lignin peroxidase (from Phanerochaete chrysosporium) and lactoperoxidase it is possible to convert (+)-valencene into nootkatone under certain conditions. In this way, the conversion is very low and is most probably due to singlet oxygen that is formed by these two enzymes (Willershausen and Graf, 1991). Several microorganisms, bacteria and fungi, were also screened for their capability to transform (+)-valencene into nootkatone all without much success. (Balfoort, 1994). Two bacteria of the genus Enterobacter from a Dutch soil and an infected local beer were isolated by enrichment cultures on (+)-valencene. These bacteria transformed (+)-valencene into nootkatone (12% yield) and many other valencene derivatives (Dhavlikar and Albroscheit, 1973). Also other methods for the bioconversion of valencene to nootkatone using inicro-organisms have been studied, but none of these methods have proven commercially viable (Dhavlikar et al., 1973; Drawert et al., 1984).